Defining how cells regulate the uptake and efflux of transition metals such as Zn and Cu is a key component in elucidating cellular mechanisms of metal homeostasis. Bacterial model systems provide paradigms for understanding metal-responsive gene regulation. In E. coli, the metalloregulator ZntR senses Zn excess and activates Zn efflux, while Zur senses Zn sufficiency and represses Zn uptake, to keep this essential metal at appropriate physiological levels in the cell. CueR, a homolog of ZntR, senses intracellular Cu to activate Cu efflux/detoxification genes to keep this toxic metal minimal. The long-term goal here is to understand how metal regulation in the cell can be manipulated for preventive and therapeutic purposes. Toward this goal, the PI has established an internationally unique research program that applies and develops advanced single- molecule single-cell approaches to interrogate and understand the mechanisms of bacterial metal regulation both in vitro and in live cells, which are further enhanced by bulk biochemical/biophysical and protein/genetic engineering approaches and established collaborations with biologists. The research has led to discoveries of first-of-their-kind mechanisms of Cu/Zn-responsive transcriptional regulation, but new questions also emerged. The objective of this renewal is to continue this program, as well as elucidate the mechanism that couples CueR/ZntR regulation to DNA mechanical tension and the mechanism of Zur?s biphasic unbinding kinetics from DNA, two novel phenomena the PI recently discovered. The premise of this research comprises the importance of (bacterial) metal regulation in biology, the discovered novel and broadly relevant regulation mechanisms, and the power of combining single-molecule/cell and bulk measurements. The proposed research contains two specific aims, each with sub-aims: 1) Identify the mechanism of DNA-mechanical-tension?coupled transcription regulation by CueR/ZntR. This aim will test hypotheses based on the discoveries that CueR/ZntR?s unbinding from DNA is modulated by chromosome condensation in cells and that CueR/ZntR can control RNAP actions on DNA. 2) Identify the mechanism of biphasic unbinding kinetics of Zur from DNA. This aim will test hypotheses regarding the preliminary results that apo/holo-Zur shows biphasic (i.e., repressed followed by facilitated) unbinding kinetics from DNA with increasing intracellular protein concentrations. The research is significant because it will elucidate novel molecular mechanisms of metalloregulators in regulating metal efflux and uptake, as well as provide fundamental knowledge about cell biology of metals in general, for identifying causes or developing preventions of diseases that involve similar regulation processes, and for helping the development of (bio)chemical strategies to manipulate bacterial Zn/Cu regulation to impair pathogen growth. The research is innovative because it applies/develops novel single-molecule manipulation, imaging, and analysis methods, and introduce new mechanistic concepts in transcription regulation.